Diphtheria toxin variant

ABSTRACT

The present invention relates to methods and compositions of modified variants of diphtheria toxin (DT) that reduce binding to vascular endothelium or vascular endothelial cells, and therefore, reduce the incidence of Vascular Leak Syndrome. One aspect of the present invention relates to a polypeptide toxophore from a modified DT, wherein the mutation is the substitution or deletion at least one amino acid residue at the amino acid residues 6-8, 28-30 or 289-291 of native DT. Another aspect of the present invention relates to a fusion protein which comprises a modified DT and a non-DT fragment. Another aspect of the present invention relates to the use of modified DT for the treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/368,254 filed Feb. 9, 2009, now issued as U.S. Pat. No.8,048,985; which is a continuation application of U.S. application Ser.No. 10/995,338 filed Nov. 24, 2004, now issued as U.S. Pat. No.7,585,942; which claims the benefit under 35 USC §119(e) to U.S.Application Ser. No. 60/524,615 filed Nov. 25, 2003. The disclosure ofeach of the prior applications is considered part of and is incorporatedby reference in the disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to methods and compositions ofmodified variants of diphtheria toxin (hereinafter “DT”) that reducebinding to vascular endothelium or vascular endothelial cells,therefore, reduce the incidence of Vascular Leak Syndrome (hereinafter“VLS”).

2. Background Information

Vascular Leak Syndrome is primarily observed in patients receivingprotein fusion toxin or recombinant cytokine therapy. VLS can manifestas hypoalbuminemia, weight gain, pulmonary edema and hypotension. Insome patients receiving immunotoxins and fusion toxins, myalgia andrhabdomyolysis result from VLS as a function of fluid accumulation inthe muscle tissue or the cerebral microvasculature [Smallshaw et al.,Nat Biotechnol. 21(4):387-91 (2003)]. VLS has occurred in patientstreated with immunotoxins containing ricin A chain, saporin, pseudomonasexotoxin A and DT. All of the clinical testing on the utility oftargeted toxins, immunotoxins and recombinant cytokines reported thatVLS and VLS-like effects were observed in the treatment population. VLSoccurred in approximately 30% of patients treated with DAB₃₈₉IL-2 [(Fosset al., Clin Lymphoma 1(4):298-302 (2001), Figgitt et al., Am J ClinDermatol, 1(1):67-72 (2000)]. DAB₃₈₉IL-2, is interchangeable referred toin this application as DT387-IL2, is a protein fusion toxin comprised ofthe catalytic (C) and transmembrane (T) domains of DT (the DTtoxophore), genetically fused to interleukin 2 (IL-2) as a targetingligand. [Williams et al., Protein Eng., 1:493-498 (1987); Williams etal., J. Biol. Chem., 265:11885-11889 (1990); Williams et al., J. Biol.Chem., 265 (33):20673-20677, Waters et al., Ann. New York Acad. Sci.,30(636):403-405, (1991); Kiyokawa et al., Protein Engineering,4(4):463-468 (1991); Murphy et al., In Handbook of ExperimentalPharmacology, 145:91-104 (2000)]. VLS has also been observed followingthe administration of IL-2, growth factors, monoclonal antibodies andtraditional chemotherapy. Severe VLS can cause fluid and proteinextravasation, edema, decreased tissue perfusion, cessation of therapyand organ failure. [Vitetta et al., Immunology Today, 14:252-259 (1993);Siegall et al., Proc. Natl Acad. Sci., 91(20):9514-9518 (1994); Balunaet al., Int. J. Immunopharmacology, 18(6-7):355-361 (1996); Baluna etal., Immunopharmacology, 37(2-3): 117-132 (1997); Bascon,Immunopharmacology, 39(3):255 (1998)].

Reduction or elimination of VLS as a side effect would represent asignificant advancement as it would improve the “risk benefit ratio” ofprotein therapeutics, and in particular, the immunotoxin and fusiontoxin subclasses of protein therapeutics. (Baluna et al., Int. J.Immunopharmacology, 18(6-7):355-361 (1996); Baluna et al.,Immunopharmacology, 37(No. 2-3):117-132 (1997); Bascon,Immunopharmacology, 39(3): 255 (1998). The ability to develop fusionproteins, single chain molecules comprised of a cytotoxin and uniquetargeting domain (scfv antibodies in the case of immunotoxins) couldfacilitate the development of the therapeutic agents for autoimmunediseases, such as rheumatoid arthritis and psoriasis transplantrejection and other non-malignant medical indications. (Chaudhary etal., Proc. Natl. Acad. Sci. USA, 87(23):9491-9494 (1990); Frankel etal., In Clinical Applications of Immunotoxins Scientific PublishingServices, Charleston S.C., (1997), Knechtle et al., Transplantation,15(63):1-6 (1997); Knechtle et al., Surgery, 124(2): 438-446 (1998);LeMaistre, Clin. Lymphoma, 1:S37-40 (2000); Martin et al., J. Am. Acad.Dermatol, 45(6):871-881, 2001)). DAB₃₈₉IL-2 (ONTAK) is currently theonly FDA approved protein fusion toxin and employs a DT toxophore andthe cytokine interleukin 2 (IL-2) to target IL-2 receptor bearing cellsand is approved for the treatment of cutaneous T-cell lymphoma (CTCL).(Figgitt et al., Am. J. Clin. Dermatol, 1(1):67-72 (2000); Foss, Clin.Lymphoma, 1(4):298-302 (2001); Murphy et al., In Bacterial Toxins:Methods and Protocols, Hoist O, ed, Humana Press, Totowa, N.J., pp.89-100 (2000)). A number of other toxophores, most notably ricin toxinand pseudomonas exotoxin A, have been employed in developing bothimmuntoxins and fusion toxins; however, these molecules have notsuccessfully completed clinical trials and all exhibit VLS as apronounced side effect (Kreitman, Adv. Pharmacol, 28:193-219 (1994);Puri et al., Cancer Research, 61:5660-5662 (1996); Pastan, BiochimBiophys Acta., 24:1333(2):C1-6 (1997); Frankel et al., Supra (1997);Kreitman et al., Current Opin. Inves. Drugs, 2(9):1282-1293 (2001)).

VLS arises from protein-mediated damage to the vascular endothelium. Inthe case of recombinant proteins, immunotoxins and fusion toxins, thedamage is initiated by the interaction between therapeutic proteins andvascular endothelial cells. Lindstrom et al. provided evidence thatricin toxin A had direct cytotoxic effects on human umbilical veinvascular epithelial cells but that these effects were not mediated byfibronectin (Lindstrom et al., Blood, 90(6):2323-34 (1997); Lindstrom etal., Methods Mol. Biol, 166:125-35 (2001)). Baluna et al. postulatedthat the interaction disrupts fibronectin mediated cell-to-cellinteractions resulting in the breakdown of vascular integrity, andBaluna further suggested that in the toxin ricin, the interaction ismediated by a conserved three amino acid motif, (x)D(y), where x is L,I, G or V and y is V, L or S (Baluna et al., Int. J. Immunopharmacology,18(6-7):355-361 (1996); Baluna et al., Proc. Natl. Acad. Sci. USA,30:96(7):3957-3962, (1999); Baluna et al., Exp Cell Res., 58(2):417-24(2000)). It was reported that one of the VLS motifs found in ricintoxin, the ‘LDV’ motif, essentially mimics the activity of a subdomainof fibronectin which is required for binding to the integrin receptor.Integrins mediate cell-to-cell and cell-to-extracellular matrixinteractions (ECM). Integrins function as receptors for a variety ofcell surface and extracellular matrix proteins including fibronectin,laminin, vitronectin, collagen, osteospondin, thrombospondin and vonWillebrand factor. Integrins play a significant role in the developmentand maintenance of vasculature and influence endothelial celladhesiveness during angiogenesis. Further, it is reported that the ricin‘LDV’ motif can be found in a rotavirus coat protein, and this motif isimportant for cell binding and entry by the virus. (Coulson, et al.,Proc. Natl. Acad. Sci. USA, 94(10):5389-5494 (1997)). Thus, it appearsto be a direct link between endothelial cell adhesion, vascularstability and the VLS motifs which mediate ricin binding to humanvascular endothelial cells (HUVECs) and vascular leak.

Mutant dgRTAs were constructed in which this motif was removed byconservative amino acid substitution, and these mutants illustratedfewer VLS effects in a mouse model (Smallshaw et al. Nat Biotechnol.,21(4):387-91 (2003)). However, the majority of these constructs yieldeddgRTA mutants that were not as cytotoxic as wild type ricin toxin,suggesting that significant and functionally critical structural changesin the ricin toxophore resulted from the mutations. It should also benoted that no evidence was provided to suggest that the motifs in dgRTAmediated HUVEC interactions and VLS in any other protein. Studiesrevealed that the majority of the mutant dgRTAs were much less effectivetoxophores and no evidence was provided to suggest that fusion toxinscould be assembled using these variant toxophores.

DT is composed of three domains: the catalytic domain; transmembranedomain; and the receptor binding domain (Choe et al. Nature, 357:216-222(1992)). Native DT is targeted to cells that express heparin bindingepidermal growth factor-like receptors (Naglish et al., Cell,69:1051-1061 (1992)). The first generation targeted toxins wereinitially developed by chemically cross-linking novel targeting ligandsto toxins such as DT or mutants of DT deficient in cell binding (e.g.CRM45). (Cawley, Cell 22:563-570 (1980); Bacha et al., Proc. Soc. Exp.Biol. Med., 181(1):131-138 (1986); Bacha et al., Endocrinology,113(3):1072-1076 (1983); Bacha et al., J. Biol. Chem., 258(3):1565-1570(1983)). The native cell binding domain or a cross-linked ligand thatdirects the DT toxophore to receptors on a specific class ofreceptor-bearing cells must possess intact catalytic and translocationdomains. (Cawley et al., Cell, 22:563-570 (1980); vanderSpek et al., J.Biol. Chem., 5:268(16):12077-12082 (1993); vanderSpek et al., J. BiolChem., 7(8):985-989 (1994); vanderSpek et al., J. Biol Chem.,7(8)985-989 (1994); Rosconi, J. Biol Chem., 10;277(19):16517-161278(2002)). These domains are critical for delivery and intoxification ofthe targeted cell following receptor internalization (Greenfield et al,Science, 238(4826)536-539 (1987)). Once the toxin, toxin conjugate orfusion toxin has bound to the cell surface receptor the cellinternalizes the toxin bound receptor via endocytic vesicles. As thevesicles are processed they become acidified and the translocationdomain of the DT toxophore undergoes a structural reorganization whichinserts the 9 transmembrane segments of the toxin into the membrane ofthe endocytic vesicle. This event triggers the formation of a productivepore through which the catalytic domain of the toxin is threaded. Oncetranslocated the catalytic domain which possess theADP-ribosyltransferase activity is released into the cytosol of thetargeted cell where it is free to poison translation thus effecting thedeath of the cell (reviewed in vanderSpek et al., Methods in MolecularBiology, Bacterial Toxins: methods and Protocols, 145:89-99, Humanapress, Totowa, N.J., (2000)).

Chemical cross-linking or conjugation results in a variety of molecularspecies representing the reaction products, and typically only a smallfraction of these products are catalytically and biologically active. Inorder to be biologically active, the reaction products must beconjugated in manner that does not interfere with the innate structureand activity of the catalytic and translocation domains in thetoxophore. Resolution of the active or highly active species from theinactive species is not always feasible as the reaction products oftenpossess similar biophysical characteristics, including for example size,charge density and relative hydrophobicity. It is noteworthy thatisolation of large amounts of pure clinical grade active product fromchemically crosslinked toxins is not typically economically feasible forthe production of pharmaceutical grade product for clinical trials andsubsequent introduction to clinical marketplace. To circumvent thisissue, a genetic DT-based protein fusion toxin in which the native DTreceptor-binding domain was genetically replaced withmelanocyte-stimulating hormone as a surrogate receptor-targeting domainwas created (Murphy et al, PNAS, 83:8258-8262 (1986)). This approach wasused with human IL-2 as a surrogate targeting ligand to createDAB₄₈₆IL-2 that was specifically cytotoxic only to those cells thatexpressed the high-affinity form of the IL-2 receptor (Williams et al.,Protein Eng., 1:493-498 (1987)). Subsequent studies of DAB₄₈₆IL-2indicated that truncation of 97 amino acids from the DT portion of themolecule resulted in a more stable, more cytotoxic version of the DL-2receptor targeted toxin, DAB₃₈₉IL-2 (Williams et al., J. Biol Chem.,265:11885-889 (1990)). The original constructs (the 486 forms) stillpossessed a portion of the native DT cell binding domain. The DAB₃₈₉amino acid residue version contains the C and T domains of DT with theDT portion of the fusion protein ending in a random coil between the Tdomain and the relative receptor binding domain. A number of othertargeting ligands have since been genetically fused to this DTtoxophore, DAB₃₈₉. (vanderSpek et al., Methods in Molecular Biology,Bacterial Toxins:Methods and Protocols., 145:89-99, Humana Press,Totowa, N.J. (2000)). Similar approaches have now been employed withother bacterial proteins and genetic fusion toxins are often easier toproduce and purify.

SUMMARY OF THE INVENTION

The present invention provides compositions of modified variants of DTthat reduce binding to vascular endothelium or vascular endothelialcells, and therefore, reduce the incidence of Vascular Leak Syndrome.

One aspect of the present invention relates to a composition comprisinga polypeptide toxophore from a DT, said polypeptide toxophore comprisingamino acid residues 7-9, 29-31 and 290-292 of SEQ ID NO:4, wherein atleast one amino acid in said amino acid residues 7-9, 29-31 or 290-292of SEQ ID NO:4 has been substituted or deleted.

Another aspect of the present invention relates to a fusion proteincomprising a modified DT mutant or fragment and a non-DT fragment.

Another aspect of the present invention relates to the use of a modifiedDT or a fusion protein carrying such modified DT for the treatment ofdiseases, such as cancer.

Yet another aspect of the present invention relates to a method ofmaking a modified DT fragment having a reduced binding activity to humanvascular endothelial cells (HUVEC) and having a reduced induction ofVascular Leak Syndrome (VLS).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of DT387. The positions of the (x)D(y)motifs of the toxophore which are implicated in VLS are indicated bytriangles and listed below in the table of proposed DT387 andcorresponding DAB₃₈₉IL2 (hereinafter “DT387IL2”) mutants. The flexiblelinker sterically enhances disulfide bond formation between theC-terminal cysteine residue and a Sulfo-LC SPDP modified targetingligand.

FIG. 2 is a ribbon diagram and space filling model of native DT showingthe presence of (x)D(y) motifs implicated in VLS,

FIG. 3 shows the nucleic acid and amino acid sequence changes in variousDT variants.

FIG. 4A shows analysis of DT387 toxophore yield by Coomassie.

FIG. 4B shows analysis of DT387 toxophore yield by Western Blot.

FIGS. 5A and 5B show representative photomicrographs illustrating thelevels of fluorescence between wild type DT toxophore mediated HUVECstaining (FIG. 5A) and VLS modified HUVEC staining (FIG. 5B).

FIG. 6 illustrates HUVEC binding to DT387 and VLS modified DT387toxophores.

FIG. 7 is a diagram showing the ADP ribosyltransferase activity ofcertain DT mutants. ADP ribosyltransferase assay in which the activityof alanine substitute VLS modified DT toxphores or DT387IL2 fusionproteins were compared to fragment A of native DT.

FIG. 8 depicts an example of Coomassie stained SDS-PAGE analysis ofcrude and purified refolded samples of DT387EGF fusion protein(DT387(D8EV29A)EGF). Tubes 1-9 not depicted.

FIG. 9 is a diagram showing the cytotoxicity of 8×10⁻⁹ MDT387(D8EV29A)linker EGF in EGF receptor positive U87MG glioblastomacells under refolding buffer conditions 1-9.

FIG. 10 is a diagram showing the cytotoxicity of 4×10⁻⁸ MDT387(D8EV29A)linkerEGF in U87MG cells under refolding buffer conditions10-18.

FIG. 11 is a diagram showing the cytotoxicity of DT389EGF and VLSmodified DT387linkerEGF fusion proteins, DT387linkerEGF,DT387(D8E,V29A)linker EGF, DEF, DT387(D8S,V29A)linkerEGF,DT387(D8E,V29A,D291E)linkerEGF, DT387(V7A,V29A)linkerEGF,DT387(V29A)linkerEGF, DT387(V7S,V29A)linkerEGF, DT387(D291E)linker IL2in U87MG cell.

FIG. 12 is a Coomassie stained SDS-PAGE gel showing the level of purityfor the VLS-modified DT387EGF fusion protein tested in FIG. 11.

FIG. 13 is a diagram showing the cytotoxicity of DT387IL2,DT387(D8S,V29A)linkerIL2, DT387(V7A,V29A,D291E)linkerIL2,DT387(V7A)linkerIL2, DT387(V7S,V29A)linkerIL2, DT387(D8E,V29A)linkerIL2in IL-2 receptor positive HUT102/6TG cells.

FIG. 14 and is a Coomassie stained SDS-PAGE gel and Western blot showingthe level of purity for the VLS-modified DT387linkerIL2 fusion proteinstested in FIG. 13.

DETAILED DESCRIPTION OF THE INVENTION

The primary objective of the present invention is to providecompositions comprising modified variants of DT that reduce binding tovascular endothelium or vascular endothelial cells, and therefore,reduce the incidence of Vascular Leak Syndrome (hereinafter “VLS”). Thesecond objective of the present invention is to provide methods ofmaking such modified variants of DT that reduce binding to vascularendothelium or vascular endothelial cells. The third objective of thepresent invention is to provide methods of treating various diseases,such as cancer, by using modified variants of DT or by using a fusionprotein comprising modified variants of DT and non-DT protein.

One aspect of the present invention relates to genetically modifiedmolecules of diphtheria toxin (DT) having reduced binding to humanvascular endothelial cells (HUVECs). These modified DT molecules arehereinafter referred to as “DT variants.” The invention specificallyrelates to DT variants having one or more conservative changes withinthe (x)D(y) motifs of the DT molecule, i.e., at residues 6-8 (VDS),residues 28-30 (VDS), and residues 289-291 (IDS) of the native DTsequence (SEQ ID NO:1), or at residues 7-9 (VDS), residues 29-31 (VDS),and residues 290-292 (IDS) of the SEQ ID NO:4. Since the (x)D(y) motifsare referred to as “VLS motifs,” the DT variants with modified (x)D(y)motif are sometimes referred to as “VLS-modified DT molecules.”

Conservative changes are defined as those amino acid substitutions whichpermit the alteration of the native sequence within these regions but donot impair the cytotoxicity of the toxophore. These conservative changeswould not include those that regenerate the VDS/IDS sequencesresponsible for mediating the interaction with endothelial cells. Suchnon-native recombinant sequences therefore comprise a novel series ofmutants that maintain the native function of the unique domains ofdiphtheria toxin while significantly decreasing their ability tointeract with vascular endothelial cells.

In one embodiment, the DT variants of the present invention contain atleast one conservative change within one of the (x)D(y) motifs of the DTmolecule, i.e., within residues 6-8 (VDS), residues 28-30 (VDS), andresidues 289-291 (IDS) of SEQ ID NO:1 to eliminate motifs that areassociated with VLS and thereby reduce the clinical adverse effectscommonly associated with this syndrome. The DT variants of the presentinvention, however, are as effective and efficient as DT387 in theirability to facilitate the delivery of its catalytic domain to thecytosol of targeted eukaryotic cells when incorporated into proteinfusion toxins. DT387 (SEQ ID NO:4) is a truncated DT protein comprisingamino acid residues 1-386 (SEQ ID NO:2) of the native DT proteinincluding the catalytic domain and the translocation domain.

In another embodiment, in addition to the modification in the (x)D(y)motifs, the DT variants may further comprise a deletion or substitutionof 1 to 30 amino acids of SEQ ID NO:4, preferably 1 to 10 amino acids,most preferably 1-3 amino acids.

To produce DT variants with a modified (x)D(y) sequence, one coulddelete or substitute another amino acid for the aspartic acid (D), orinsert one or more amino acids at or adjacent to its position. Any aminoacid that may replace the (D) residue in the sequence as a consequenceof a deletion or mutation event must retain the ability to effectivelydeliver the catalytic domain of DT to a targeted cell within the contextof a fusion protein, and not reconstitute an intact VLS motif.

Alternatively the (x) residue could be deleted, substituted, or moved bythe insertion of one or more amino acids, to remove the (x)D(y)sequence. Any amino acid that may replace the (x) residue in thesequence as a consequence of the deletion or mutation event shouldpreferably not be leucine (L), isoleucine (I), glycine (G) or valine(V). The (y) residue could be deleted, substituted, or moved by theinsertion of one or more amino acids, to remove the (x)D(y) sequence.Any amino acid that may replace the (y) residue in the sequence as aconsequence of the deletion or mutation event should preferably not bevaline (V), leucine (L) or serine (S).

In a preferred embodiment, the DT variants of the present inventioncontain at least one of the mutations selected from the group of V7A,V7S, DBS, D8E, V29A, I290A, D291S, and D291E. It should be noted thatthe first amino acid residue of mature processed native DT proteincorresponds to the second amino acid residue of the DT variants(recombinant expression requires insertion of met residue). Accordingly,residues 6-8 (VDS), 28-30 (VDS) and 289-291 (IDS) of the native DTcorrespond to residues 7-9, 29-31, and 290-292 of the DT variants.

In another preferred embodiment, the DT variants of the presentinvention contain a double mutation selected from the group of V7AV29A,V7SV29A, D8SV29A, D8SD291S, D8EV29A, and V29AD291E.

In another preferred embodiment, the DT variants of the presentinvention contain a triple mutation selected from the group ofV7AV29AD291E and V7AV29AI290A.

In yet another preferred embodiment, the DT variants comprise an aminoacid sequence recited in one of SEQ ID NOs:28-38. It is conceivable thatother residues that are positioned in the physical region,three-dimensional space, or vicinity of the HUVEC binding site and/orthe (x)D(y) motif may be mutated or altered to abrogate, reduce, oreliminate VLS. The amino acids targeted for mutation in the flankingregions include amino acids on or near the surface of a native DTprotein. The alteration may remove or substitute a charged residue inthe region of a (x)D(y) motif, which may negate or reverse the charge ina particular area on the surface of the protein. The alteration may alsochange size and/or hydrophilic nature of an amino acid in the physicalregion, space or vicinity of the (x)D(y) sequence or active site of aprotein.

In certain aspects, mutagenesis of nucleic acids encoding peptides,polypeptides or proteins may be used to produce the desired mutations inthe (x)D(y) and flanking sequences of the DT variants. Mutagenesis maybe conducted by any means disclosed herein or known to one of ordinaryskill in the art.

One particularly useful mutagenesis technique is alanine scanningmutagenesis in which a number of residues are substituted individuallywith the amino acid alanine so that the effects of losing side-chaininteractions can be determined, while minimizing the risk of large-scaleperturbations in protein conformation [(Cunningham et al., Science,2:244(4908): 1081-5 (1989)].

As specific amino acids may be targeted, site-specific mutagenesis is atechnique useful in the preparation of individual peptides, orbiologically functional equivalent proteins or peptides, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of themutation site being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art. Briefly, a bacteriophage vector that will produce a singlestranded template for oligonucleotide directed PCR mutagenesis isemployed. These phage vectors, typically M13, are commercially availableand their use is generally well known to those skilled in the artSimilarly, double stranded plasmids are also routinely employed in sitedirected mutagenesis, which eliminates the step of transferring the geneof interest from a phage to a plasmid. Synthetic oligonucleotide primersbearing the desired mutated sequence are used to direct the in vitrosynthesis of modified (desired mutant) DNA from this template and theheteroduplex DNA used to transform competent E. Coli for the growthselection and identification of desired clones. Alternatively, a pair ofprimers may be annealed to two separate strands of a double strandedvector to simultaneously synthesize both corresponding complementarystrands with the desired mutation(s) in a PCR reaction.

In one embodiment, the Quick Change site-directed mutagenesis methodusing plasmid DNA templates as described by Sugimoto et al. is employed(Sugimoto et al, Annal Biochem., 179(2):309-311 (1989)). PCRamplification of the plasmid template containing the insert target geneof insert is achieved using two synthetic oligonucleotide primerscontaining the desired mutation. The oligonucleotide primers, eachcomplementary to opposite strands of the vector, are extended duringtemperature cycling by mutagenesis-grade PfuTurbo DNA polymerase. Onincorporation of the oligonucleotide primers, a mutated plasmidcontaining staggered nicks is generated. Amplified un-methylatedproducts are treated with Dpn I to digest methylated parental DNAtemplate and select for the newly synthesized DNA containing mutations.Since DNA isolated from most E. Coli strains is dam methylated, it issusceptible to Dpn I digestion, which is specific for methylated andhemimethylated DNA. The reaction products are transformed into highefficiency strains of E. coli to obtain plasmids containing the desiredmutants.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants. These basic techniques, the protocols for sequencedetermination, protein expression and analysis are incorporated byreference to citations in this specification and are generallyaccessible to those reasonably skilled in the art within CurrentProtocols in Molecular Biology (Fred M. Ausubel, Roger Brent, Robert E.Kingston, David D. Moore, J. G. Seidman, John A. Smith, Kevin Struhl,Editors John Wiley and Sons Publishers (1989)).

The present invention also provides DT fusion proteins. A DT fusionprotein contains a DT-related polypeptide (e.g., a DT variant of thepresent invention) and a non-DT polypeptide fused in-frame to eachother. The DT-related polypeptide corresponds to all or a portion of DTvariant having reduced binding to human vascular endothelial cells. Inone embodiment, a DT fusion protein comprises at least one portion of aDT variant sequence recited in one of SEQ ID NOs:11-27.

Preferably, a DT-fusion protein of the present invention is produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example, by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence.

A peptide linker sequence may be employed to separate the DT-relatedpolypeptide from non-DT polypeptide components by a distance sufficientto ensure that each polypeptide folds into its secondary and tertiarystructures. Such a peptide linker sequence is incorporated into thefusion protein using standard techniques well known in the art. Suitablepeptide linker sequences may be chosen based on the following factors:(1) their ability to adopt a flexible extended conformation; (2) theirinability to adopt a secondary structure that could interact withfunctional epitopes on the DT-related polypeptide and non-DTpolypeptide; and (3) the lack of hydrophobic or charged residues thatmight react with the polypeptide functional epitopes. Preferred peptidelinker sequences contain gly, asn and ser residues. Other near neutralamino acids, such as thr and ala, may also be used in the linkersequence. Amino acid sequences which may be usefully employed as linkersinclude those disclosed in Maratea et al., Gene, 40:39-46, 1985; Murphyet al., Proc. Natl. Acad. Sci. USA, 83:8258-8262, 1986; U.S. Pat. No.4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generallybe from 1 to about 50 amino acids in length. Linker sequences are notrequired when the DT-related polypeptide and non-DT polypeptide havenon-essential N-terminal amino acid regions that can be used to separatethe functional domains and prevent steric interference. For example,DT389/DT387-linker has a sequence of SEQ ID NO:5, DT380-linker hassequence of SEQ ID NO:6. The non-DT polypeptides can be any polypeptide.

In one embodiment, the non-DT polypeptide is a cell-specific bindingligand. The specific-binding ligands used in the invention can containan entire ligand, or a portion of a ligand which includes the entirebinding domain of the ligand, or an effective portion of the bindingdomain. It is most desirable to include all or most of the bindingdomain of the ligand molecule.

Specific-binding ligands include but not limited to: polypeptidehormones, chimeric toxins, e.g., those made using the binding domain ofα-MSH, can selectively bind to melanocytes, allowing the construction ofimproved DT-MSH chimeric toxins useful in the treatment of melanoma.(Murphy, J. R., Bishai, W., Miyanohara, A., Boyd, J., Nagle, S., Proc.Natl. Acad. Sci. U.S.A., 83(21):8258-8262 (1986)). Otherspecific-binding ligands which can be used include insulin,somatostatin, interleukins I and HI, and granulocyte colony stimulatingfactor. Other useful polypeptide ligands having cell-specific bindingdomains are follicle stimulating hormone (specific for ovarian cells),luteinizing hormone (specific for ovarian cells), thyroid stimulatinghormone (specific for thyroid cells), vasopressin (specific for uterinecells, as well as bladder and intestinal cells), prolactin (specific forbreast cells), and growth hormone (specific for certain bone cells).Specific-binding ligands which can be used include cytokines. Examplesof cytokines include, but are not limited to, IL-1, IL-2, IL-3, IL-4,EL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,P-interferon, α-interferon (INFα), INFγ, angiostatin, thrombospondin,endostatin, METH-1, METH-2, GM-CSF, G-CSF, M-CSF, tumor necrosis factor(TNF), SVEGF, TGFβ, Flt3 and B-cell growth factor. IL-2 is of particularimportance because of its role in allergic reactions and autoimmunediseases such as systemic lupus erythmatosis (SLE), involving activatedT cells. DT fusion protein made using B-cell growth factor can be usedas immunosuppressant reagents which kill proliferating B-cells, whichbear B-cell growth factor receptors, and which are involved inhypersensitivity reactions and organ rejection. Other preferredcytokines include Substance P (Benoliel et al., Pain, 79(2-3):243-53(1999)), VEGF (Hotz et al., J Gastrointest Surg., 6(2):159-66 (2002)),IL3 (Jo et al., Protein Exp Purif. 33(1):123-33 (2004)) and GMCSF(Frankel et al., Clin Cancer Res, 8(5):1004-13 (2002)). VLS modified DTfusion toxins using these ligands are useful in treating cancers orother diseases of the cell type to which there is specific binding.

For a number of cell-specific ligands, the region within each suchligand in which the binding domain is located is now known. Furthermore,recent advances in solid phase polypeptide synthesis enable thoseskilled in this technology to determine the binding domain ofpractically any such ligand, by synthesizing various fragments of theligand and testing them for the ability to bind to the class of cells tobe labeled. Thus, the chimeric genetic fusion toxins of the inventionneed not include an entire ligand, but rather may include only afragment of a ligand which exhibits the desired cell-binding capacity.Likewise, analogs of the ligand or its cell-binding region having minorsequence variations may be synthesized, tested for their ability to bindto cells, and incorporated into the hybrid molecules of the invention.Other potential ligands include antibodies (generally monoclonal) orantigen-binding, single-chain analogs of monoclonal antibodies, wherethe antigen is a receptor or other moiety expressed on the surface ofthe target cell membrane. The antibodies most useful are those againsttumors; such antibodies are already well-known targeting agents used inconjunction with covalently bound cytotoxins. In the present invention,the anti-tumor antibodies (preferably not the whole antibody, but justthe Fab portion) are those which recognize a surface determinant on thetumor cells and are internalized in those cells via receptor-mediatedendocytosis; antibodies which are capped and shed will not be aseffective.

Another aspect of the present invention pertains to vectors containing apolynucleotide encoding a DT variant or a DT fusion protein. One type ofvector is a “plasmid,” which includes a circular double-stranded DNAloop into which additional DNA segments can be ligated. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of vectors, such as expressionvectors, and gene delivery vectors.

The expression vectors of the invention comprise a polynucleotideencoding DT variant or a DT fusion protein in a form suitable forexpression of the polynucleotide in a host cell. The expression vectorsgenerally have one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the polynucleotide sequence to be expressed. It will be appreciatedby those skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to produceproteins or peptides, including fusion proteins or peptides, encoded bypolynucleotides as described herein (e.g., a DT variant or a DT fusionprotein, and the like)

The expression vectors of the present invention can be designed forexpression of a DT variant or a DT fusion protein in prokaryotic oreukaryotic cells. It should be noted that the presence of a single DTmolecule inside an eukaryotic cell would kill the cell. Specifically,the toxin binds to EF-tu which is required for translation andribosylation. Accordingly, DT can only be expressed in cells withmodified EF-tu that is no longer recognized by DT. (see, e.g., Liu etal., Protein Expr Purif., 30:262-274 (2003); Phan et al., J. Biol Chem.,268(12):8665-8 (1993); Chen et al., Mol. Cell Biol, 5(12):3357-60(1985); Kohne et al., Somat Cell Mol Genet., 11(5):421-31 (1985);Moehring et al., Mol. Cell Biol., 4(4):642-50 (1984)). In addition, a DTvariant or a DT fusion protein can be expressed in bacterial cells suchas E. coli [Bishai et al., J Bacteriol 169(11):5140-51 (1987)].Consideration must be given to the expression and activity of the typesand levels of host protease expression, and this is dependent upon thecleavage site present in the engineered DT toxophore. The innateexpression host protease expression profile could negatively impact theyields of DT fusion toxin produced [Bishai et al., Supra (1987)]. To thedegree that this requisite cleavage site can be altered to modulate thecell selectivity of resultant fusion proteins, it is envisioned thatsuch cleavage site mutants could be in VLS-modified toxophores (Gordonet al., Infect Immun, 63(1):82-7 (1995); Gordon et al., Infect Immun,62(2):333-40 (1994); Vallera et al., J Natl. Cancer Inst., 94:597-606(2002); Abi-Habib et al., Blood., 104(7):2143-8 (2004)]. Alternatively,the expression vector can be transcribed and translated in vitro.

The present invention further provides gene delivery vehicles for thedelivery of polynucleotides to cells, tissue, or a mammal forexpression. For example, a polynucleotide sequence of the presentinvention can be administered either locally or systemically in a genedelivery vehicle. These constructs can utilize viral or non-viral vectorapproaches in in vivo or ex vivo modality. Expression of such codingsequences can be induced using endogenous mammalian or heterologouspromoters. Expression of the coding sequence in vivo can be eitherconstitutive or regulated. The invention includes gene delivery vehiclescapable of expressing the contemplated polynucleotides including viralvectors. For example, Qiao et al., developed a system employing PG13packaging cells produce recombinant retroviruses carrying a DT fragmentwhich kills cancer cell and provides a method for using DT as componenta suicide vector. Qiao et al., J. Virol. 76(14):7343-8 (2002).

Expressed DT-mutants and DT-fusion proteins can be tested for theirfunctional activity. Methods for testing DT activity are well-known inthe art. For example, the VLS effect of DT-mutants and DT-fusionproteins can be tested in HUVECs as described in Example 2. Theribosyltransferase activity of DT variants or DT-fusion proteins can betested by the ribosyltransferase assay described in Example 3. Thecytotoxicity of DT variants or DT-fusion proteins can be tested asdescribed in Examples 4-5.

DT-mutants and DT-fusion proteins having reduced binding to HUVECs whilemaintaining the cytotoxicity can be used for the treatment of variouscancers, including, but not limited to breast cancer, colon-rectalcancer, lung cancer, prostate cancer, skin cancer, osteocarcinoma, orliver cancer and others.

In an exemplary embodiment, the VLS modified DT fusion toxins of theinvention are administered to a mammal, e.g., a human, suffering from amedical disorder, e.g., cancer, or non-malignant conditionscharacterized by the presence of a class of unwanted cells to which atargeting ligand can selectively bind.

The pharmaceutical composition can be administered orally or byintravenously. For example, intravenous now possible by cannula ordirect injection or via ultrasound guided fine needle. Mishra (Mishra etal., Expert Opin. Biol, 3(7):1173-1180 (2003)) provides for intratumoralinjection.

The term “therapeutically effective amount” as used herein, is thatamount achieves at least partially a desired therapeutic or prophylacticeffect in an organ or tissue. The amount of a modified DT necessary tobring about prevention and/or therapeutic treatment of the disease isnot fixed per se. The amount of VLS modified DT fusion toxinadministered will vary with the type of disease, extensiveness of thedisease, and size of species of the mammal suffering from the disease.Generally, amounts will be in the range of those used for othercytotoxic agents used in the treatment of cancer, although in certaininstances lower amounts will be needed because of the specificity andincreased toxicity of the VLS-modified DT fusion toxins. In certaincircumstances and as can be achieved by currently available techniquesfor example (cannulae or convection enhanced delivery, selectiverelease) attempts to deliver enhanced locally elevated fusion toxinamounts to specific sites may also be desired. (Laske et al., JNeurosurg., 87:586-5941(997); Laske et al., Nature Medicine, 3:1362-1368(1997), Rand et al., Clin. Cancer Res., 6:2157-2165 (2000); Engebraatenet al., J. Cancer, 97:846-852 (2002), Prados et al, Proc. ASCO, 21:69b(2002), Pickering et al., J Clin Invest, 91(2):724-9 (1993)).

The invention is further directed to pharmaceutical compositionscomprising a DT variant or DT-fusion protein described hereinabove and apharmaceutically acceptable carrier.

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, solubilizers, stabilizers,absorbents, bases, buffering agents, lubricants, controlled releasevehicles, diluents, emulsifying agents, humectants, lubricants,dispersion media, coatings, antibacterial or antifungal agents, isotonicand absorption delaying agents, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well-known in the art. See e.g., A. H. KibbeHandbook of Pharmaceutical Excipients, 3rd ed. Pharmaceutical PressLondon, UK (2000). Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary agents can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, or glycerine;propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfate; chelating agents such as emylene-diaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose pH whichcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Mainly if not exclusively this pharmaceutical compositions suitable forinjectable use include sterile aqueous solutions (where water soluble)or dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. For intravenousadministration, suitable carriers include physiological saline,bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the injectablecomposition should be sterile and should be fluid to the extent thateasy syringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption (e.g.aluminum monostearate or gelatin), however, any stabilizer or additiveposited by this disclosure envisioned for use in protein fusion toxindelivery will be compatible with protein based therapeutics.

Sterile injectable solutions can be prepared by incorporating the activeingredient (e.g., a viral or non viral vector) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the activeingredient into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Toxicity and therapeutic efficacy of such ingredient can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage tononcancerous and otherwise healthy cells and, thereby, reduce sideeffects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration arrange that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture and as presented below examples 4-5. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography. The pharmaceutical compositions canbe included in a container, pack, or dispenser together withinstructions for administration.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables, are incorporatedherein by reference.

EXAMPLE 1 Construction, Expression and Purification of DT Variant andDT-Fusion Proteins

1(a) Construction of DT Variant and DT-Fusion Proteins

A truncated DT-based toxophore comprising a methionine residue at theN-terminus, amino acid residues 1 through 386 (SEQ ID NO:2) of thenative DT (now residues 2-387 in the truncated toxophore), and twoadditional amino acids residues His and Ala at the C-terminal wasconstructed. The inclusion of the His and Ala residues was resulted fromadditional nucleotide sequences introduced during the cloning process.This construct is designated as DT387 (SEQ ID NO:4). A schematic diagramof DT387 is shown in FIG. 1 which is equivalent to the DAB389 constructdescribed by Williams et al. with the exception that codon was alteredand optimized for E. Coli rather than C. diphtheria. A similar constructcontaining amino acid residues 1 through 379 of the native DT, with amethionine residue at the N-terminal, was also constructed, which wasdesignated was DT380 (SEQ ID NO:3). A DT380 variant with a linker andcarboxy terminal cysteine was used to determine the effects of VLSmutations on ribosyltransferase activities and to determine propensityto induce VLS as a function of HUVEC binding.

As shown in FIG. 2, native DT contains three (x)D(y) motifs at residues6-8 (VDS), residues 28-30 (VDS), and residues 289-291 (IDS). (The numberof the residues in the genetic constructs is +1 with respect to nativeDT). Briefly, site directed mutagenesis was employed to alter the(x)D(y) motif in DT387. A Stratagene Quickchange mutagenesis kit wasused to construct the mutations. Oligonucleotide primers were designedto alter encoding residues within the (x)D(y) motif implicated in VLS.

Table 1 provides a list of all the DT mutants that were created,expressed in E. coli, partially purified (not to absolute homogeneity)and tested for cytotoxicity. The corresponding nucleic acid and aminoacid sequence changes are shown in FIG. 3. The mutants were tested inthe context of protein fusion toxin genetically fused to sequencesencoding either human interleukin 2 or human epidermal growth factor.

TABLE 1 Mutant DT Toxophores SINGLE MUTANT SEQ ID NOs DT387(V7A) 28DT387(D8S) 29 DT387(ΔD8) 44 DT387(V7S) 30 DT387(D8E) 31 DT387(V29A) 32DT387(V290A) 33 DT387(D291E) 34 DOUBLE MUTANT SEQ ID NO DT387(V7A, V29A)35 DT387(V7S, V29A) 36 DT387(D8E, V29A) 37 DT387(D8S, V29A) 38DT387(V29A, D291E) 39 TRIPLE MUTANT SEQ ID NO DT387(V7A, V29A, I290A) 40DT387(V7A, V29A, D291E) 41

A number of DT-fusion proteins were also expressed and purified. Theseproteins and their corresponding DT counterparts are listed in Table 2.

TABLE 2 DT-Fusion Proteins and Control Proteins. SEQ ID NO FusionProteins DT387EGF/DAB389 EGF 7 DT387 linker EGF/DAB389 linker EGF 8DT387IL2/DAB389IL-2 9 DT387 linker IL2/DAB389 linker IL2 10 DT387(V7A)linkerIL2 11 DT387(D8S) linker IL2 12 DT387(D8E) linker IL2 13DT387(V29A) linker IL2 14 DT387(I290A) linker IL2 15 DT387(D291E) linkerIL2 16 DT387(V7AV29A) linker IL2 17 DT387(V7AV29AD291E) linker IL2 18DT387(D8SV29A) linker IL2 19 DT387(V7SV29A) linker IL2 42 DT387(D8EV29A)linker IL2 43 DAB389(V7AV29AI290A) linker IL2 20 DT387(V29A) linker EGF21 DT387(D291E) linker EGF 22 DT387(D8EV29A) linker EGF 23DT387(V7SV29A) linker EGF 24 DT387(V7AV29A) linker EGF 25DT387(D8EV29AD291E) linker EGF 26 DT387(D8SV29A) linker EGF 27DT387(V7A) IL2 46 DT387(D8S) IL2 47 DT387(D8E) IL2 48 DT387(V29A) IL2 49DT387(I290A) IL2 50 DT387(D291E) IL2 51 DT387(V7AV29A) IL2 52DT387(V7AV29AD291E) IL2 53 DT387(D8SV29A) IL2 54 DT387(V7SV29A) IL2 55DT387(D8EV29A) IL2 56 DAB389(V7AV29AI290A) IL2 57 DT387(V29A) EGF 58DT387(D291E) EGF 59 DT387(D8EV29A) EGF 60 DT387(V7SV29A) EGF 61DT387(V7AV29A) EGF 62 DT387(D8EV29AD291E) EGF 63 DT387(D8SV29A) EGF 64Corresponding DT Counterparts DT387/DAB389 41(b) Expression and Purification of DT Variants and DT-Fusion Proteins

Plasmid constructs encoding truncated DT protein, DT mutants, andDT-fusion protein were transformed into E. coli HMS 174 (DE3) cells. E.coli HMS 174 is a protease-deficient strain in which over-expression ofrecombinant proteins can be achieved. Induction of the recombinantprotein expression was obtained by addition ofisopropylthiogalactosidase (ITPG) to E. coli HMS 174. Followingincubation, the bacterial cells were harvested by centrifugation andlysed, and the recombinant protein was further purified from inclusionbody preparations as described by Murphs and vanderSpek, Methods inMolecular Biology, Bacterial Toxins methods and protocols, 145:89-99Humana press, Totowa, N.J. (2000). The crude protein preparations werecontaminated with endotoxin levels of between 2.5×10⁴ and 2.5×10⁵ EU/ml.It was necessary to remove endotoxins from the protein preparations toassure that effects on HUVECs are from VLS and not due to the presenceof the endotoxins. Endotoxin was removed to <250 EU/ml by passage overan ion-exchange resin. As shown in FIG. 4, separation of breakdownproducts from full-length material also occurred during the ion-exchangechromatography. After another final purification over ion exchange resinendotoxin was reduced to <25 EU/nil and the toxophore was tested for VLSas a function of HUVEC cell binding in vitro. FIG. 4 is the analysis ofDT387 toxophore yield by Coomassie and Western Blot. Samples from pilotproduction process described above resolved by SDS Polyacrylamide GelElectrophoresis (PAGE). Samples 7 through 13 elute from column with lessthan 250 EU/ml [initial levels>25,000 EU/ml] and as essentially pure DTtoxophore. Molecular weight standards are indicated (kDa). An anti-DTantibody was used for the Western blot.

Some of the constructs are more difficult to express and purify.Mutations that result in stable constructs with adequate expression thatdo not affect ribosyltransferase activity of the DT387 toxophore weresubsequently tested for targeted cytotoxicity in the corresponding VLSmodified DT-EGF and VLS modified DT-IL-2 protein fusion toxins (Examples4 and 5 respectively).

As described in more detail in Examples 2-4, DT387, VLS modifiedDT387EGF and DT387EL-2 have been used to distinguish between effects ofthe VLS mutations on catalytic activity, VLS activity and effectivedelivery of the targeted protein fusion toxins to the cytosol of targetcells.

EXAMPLE 2 Binding of DT Toxophores to HUVEC In Vitro

Human vascular endothelial cells were maintained in EGM media (obtainedfrom Cambrex, Walkersville, Md.). Sub-confluent early passage cells wereseeded at equivalent cell counts onto plastic cover slips. Purified,endotoxin free wild type DT toxophore and mutants DT38(V7AV29A)gscys andDT380(D8SD291 S)gscys were labeled with the fluorescent tag F-150(Molecular Probes, Eugene, Oreg.) through chemical conjugation. HUVECswere incubated with equivalent amounts of the labeled toxophores. Themedia was then aspirated, the cells washed and then, fixed and preparedfor analysis. Examination of the cells on cover slips from differenttreatment groups permitted the analysis of the number of cells labeledby the fluorescent toxophore. No targeting ligand was present on thetoxophore, and consequently, the level of HUVEC interaction wasproportional only to the toxophores affinity for HUVECs. Comparisonswere carried out using a fluorescent microscope and comparing the numberof cells labeled from at least ten independent fields, differentcoverslips or different slids. DAPI stain was used to localize cells,particularly in the case of the mutant constructs as cell labeling wasnot readily apparent. 4′-6-Diamidino-2-phenylindole (DAPI) is known toform fluorescent complexes with natural double-stranded DNA, as suchDAPI is a useful tool in various cytochemical investigation. When DAPIbinds to DNA, its fluorescence is strongly enhanced. Thus, DAPI servesas a method of labeling cell nuclei. In contrast, cells treated withF-150DT toxophore were easily observed. To facilitate thatquantification of the mutant DT toxophore constructs the signalintensity and change in background signal were also increased.

FIGS. 5A and 5B show representative photomicrographs illustrating thelevels of fluorescence between wild type DT toxophore mediated HUVECstaining and VLS modified HUVEC staining. There is a discernabledifference in the number of cells labeled and the intensity of thelabeled cells using the native DT toxophore molecule versus the VLSmodified molecules. As shown in FIG. 6, this change in labeling accountsfor the greater than ten fold decrease in average fluorescence observedwhen a VLS modified DT toxophore is employed to label HUVECs.

EXAMPLE 3 VLS Mutants Retain ADP-Ribosyltransferase Activity

Ribosome inactivating protein toxins such as diphtheria toxin catalyzethe covalent modification elongation factor to (EF-tu). Ribosylation ofa modified histidine residue in EF-tu halts protein synthesis at theribosome and results in cell death. Ribosyltransferase assays todetermine catalytic activity of the DT387 mutants are performed in 50 mMTris-Cl, pH8.0, 25 mM EDTA, 20 mM Dithiothreitol, 0.4 mg/ml purifiedelongation factor tu, and 1.0 pM [³²P]-NAD⁺ (10 mCml, 1000 Ci.mmol,Amersham-Pharmacia). The purified mutant proteins are tested in a finalreaction volume of 40 μl. The reactions are performed in 96 well,V-bottom microtiter plates (Linbro) and incubated at room temperaturefor an hour. Proteins are precipitated by addition of 200 μl 10% TCA andcollected on glass fiber filters, and radioactivity dis etermined bystandard protocols.

As shown in FIG. 7, DT mutants DT387(V7A), DT387(V7A)linkerIL-2,DT387(V7AV29A), and DT387(V7AV29A)linkerIL-2 exhibited ADPribosyltransferase activity that was equivalent or higher than thatexhibited by fragment A of native DT.

EXAMPLE 4 VLS Mutants Suitable for DT-Fusion Proteins-VLS-Modified DTEGF

The ability to create, express and obtain selective receptor-specificfusion toxins using VLS modified DT-based toxophores is central to thedisclosure. Proteins expressed from gene fusions between the modifiedtoxophore and a specific targeting ligand allow the development offusion toxins that can be used as research tools, as in vitro componentsof developing cell therapies and in vivo as therapeutics.

Cells integrate a variety of signals required for the maintenance ofhomeostasis and normal tissue function. These signals include solublefactors liberated by adjacent cells, regional tissue-specific factorsand signals from remote sites with an organism. These signals can takethe form of proteins, cytokines, hormones, peptides, enzymes,metabolites or small signaling molecules. The target cells expressreceptors specific to these signaling molecules and these receptors arecritical for the appropriate reception and integration of these signals.Diseases such as cancer are often characterized by aberrant signalingand some signals have been shown to stimulate proliferation anddifferentiation of the malignant cells. The Epidermal Growth Factor, orEGF is a peptide cytokine that plays a variety of roles in the bodyincluding a role as a proliferation factor for cells bearing the EGFreceptor or receptors capable of binding EGF. Inappropriate signalingthrough EGF receptors has been implicated in a number of tumorsincluding breast cancer, squamous cell cancer of the head and neck,pancreatic cancer and glioblastoma. In the case of glioblastoma,patients often exhibit a rearrangement of the gene encoding the EGFreceptor in tumor tissue. The rearrangement is typically associated witha dramatic over-expression of this growth factor receptor on thecancerous cells. This differential expression of EGF receptor on tumorcells makes it possible to direct an EGF diphtheria toxin protein fusiontoxin to these cancerous cells and selectively ablate them from thepatient. (Shaw, et al., Jour. Biol. Chem., 266:21118-21124 (1991)) EGFsignaling has also been implicated in the establishment of new bloodvessel formation are process known as angiogenesis. Angiogenesis isimportant in the development of a number of tumors and thus, DTEGFfusion toxin could be employed to prevent angiogeneisis in solid tumorsand reduce its size or prevent its development. Thus a VLS-modifiedDTEGF would have clinical utility and could be used to treat a number ofdiseases characterized by aberrant EGF receptor expression.

In addition there are circumstances in which normally appropriate EGFsignaling is undesirable and the use of a DTEGF fusion toxin under thesecircumstance could be clinically useful. For example, as described byPickering et al “smooth muscle cell proliferation in arteries is acommon event after balloon angioplasty and bypass surgery and it isassociated with vascular narrowing”. DTEGF can be utilized to preventsmooth muscle cell proliferation and it can be locally applied toprevent vascular narrowing. (Pickering et al., J Clin Invest91(2):724-9(1993)).

To determine if the modified VLS DT-based toxophore described abovecould be employed to create viable, active fusion toxins, VLS-modifiedDT387linkerEGF fusion toxins were created and tested. Plasmids encodingVLS-modified toxophores were used as starting vectors and an in-frameinsertion of the nucleotide sequence encoding EGF was inserted to createVLS-modified DTE387linkerEGF fusion proteins.

The fusion proteins were expressed essentially as described above.Induction of mutant DT387linker EGF fusion protein expression wasobtained by addition of isopropylthiogalactosidase (ITPG) to E. coliHMS174 (DE3). E. coli HMS174 is a protease-deficient strain in whichover-expression of recombinant proteins can be achieved. Followingincubation, the bacterial cells were harvested by centrifugation, theDTEGF bacterial pellets were homogenized in 20 ml, ice cold, STET buffer(50 mM Tris-Cl, pH 8.0, 10 mM EDTA, 8% glucose, 5% Triton X-100).Lysozyme was added to 25 μg/ml and the bacteria were incubated on icefor 1 hour. The preparation was homogenized and then subjected tocentrifugation at 6000×g for 30 minutes to 4° C. The resulting pelletwas resuspended in 20 ml of STET and homogenized and the centrifugationstep repeated. The final pellet was resuspended in 5 ml 7M GuHCl, 50 mMTris-Cl, pH 8.0, homogenized and centrifuged, 6000×g, 30 minutes, 4° C.The supernatant was used in refolding assays.

The supernatant protein concentration was 5 mg/ml and refolding wasperformed at final concentrations of 0.4 mg/ml and 0.08 mg/ml. Refoldingwas assessed using a Pro-Matrix protein refolding kit from Pierce.(Pierce Biotechnology Inc., Rockford, Ill.) The refolding conditions areshown in Table 3.

TABLE 3 Folding Conditions in Pierce Pro-Matric Refolding Kit Used toDetermine Optimal Refolding Conditions for VLS-Modified DT387linker EGFFusion Proteins Produced in E. coli. GuHCl L-Arginine GSG GSSG Tubes (M)(M) (mM) (mM) 1 + 10 0 0 2 0.2 2 + 11 0 0.4 2 0.4 3 + 12 0 0.8 1 1 4 +13 0.5 0 2 0.4 5 + 14 0.5 0.4 1 1 6 + 15 0.5 0.8 2 0.2 7 + 16 1.0 0 1 18 + 17 1.0 0.4 2 0.2 9 + 18 1.0 0.8 2 0.4

Tubes 1-9 had a final concentration of 1.6×10⁻⁶ M and tubes 10-18 had afinal concentration of 8×10⁻⁶ M DT387 (D8EV29A)linker EGF. The tubeswere incubated overnight at 4° C. and 1 μl volumes were assayed the nextday for cytotoxicity on U87MG glioblastoma cells (Glioblastoma cellshave been shown to express EGF receptors ((Frankel et al., Clin CancerRes., 8(5): 1004-13 (2002)). The samples were also analyzed by gelelectrophoresis to assure no degradation had occurred during refolding(FIG. 8).

Samples of VLS-modified fusion toxins (shown here VLS-modifiedDT387(D8EV29S)linker EGF and DT387EGF were purified through theinclusion body step, denatured and subjected to refolding under avariety of conditions. Samples were then dialysed to remove any residualcontaminants from the refolding conditions and tested for activity incytotoxicity assays against U87MG EGF-receptor-bearing cells. Thesepreparations are still considered crude and were used only to compareconditions which resulted in enhanced activity relative to standardrefolding conditions and fusion toxins created using the native DTtoxophore [in the context of an EGF fusion toxin DTEGF]. FIG. 9 showsthe results for cytotoxicity assays of samples 1-9, using proteinconcentration of DT387(D8EV29A)linker EGF of 8×10⁻⁹ M. FIG. 10 depictsthe results for tubes 10-18 in which the protein concentration ofDT387(D8EV29A)linker EGF employed were higher (4×10⁻⁸ M). Refolding toan actively cytotoxic form was more efficient when the lowerconcentrations of DT387(D8EV29A)linker EGF fusion toxin were employed.Buffer conditions 4, 6 and 8 were chosen for further refinement.

New preparations of DT387EGF and DT387(D8EV29A)linker EGF were preparedas described above. The final, denatured supernatants were refolded inbuffers 4, 6 or 8, (see Table 3), at lower protein concentrations. Afterrefolding, the samples were dialyzed against corresponding refoldingbuffers, without GuHCl, permitting higher concentrations of fusion toxinto be tested. The results indicate that the IC_(50S) for DT387EGF rangedfrom 8×10⁻¹⁰ M to 1.5×10⁻⁹M for the buffers tested. Buffer 8 appeared toyield the most productive protein. The same holds true for refolding ofthe DT387(D8EV29A)linker EGF mutant.

Other VLS-modified DT387EGF fusion proteins were also tested for theircytotoxicity in EGF-receptor-positive U87MG glioblastoma cells. As shownin FIG. 11, the EGF fusion toxins created with VLS modifications thatexhibit the greatest selective toxicity against EGF-receptor-bearingcell are DT387(V7SV29A)linker EGF, DT387(D291E)linker EGF,DT387(V29A)linker EGF. These EGF fusion toxins display IC 50s comparableto cGMP prepared DT387linker EGF and DT 387linker EGF prepared underconditions identical to those used to express, refold and purify theVLS-modified DT387linker EGF fusion toxins. Thus, the VLS modifiedDT-based toxophores can be employed to create novel DT-based fusiontoxins.

FIG. 12 is a Coomassie stained SDS-PAGE gel showing the level of purityfor the VLS-modified DT387linker EGF fusion protein tested in FIG. 11.Several additional species are apparent on the gel and the variation ofexpression levels can be observed. Resolution of these species by bothanion exchange chromatography and sizing yielded fusion preparationsthat were homogenous and exhibited higher specific toxicity. Thispresumably was a function of the relatively purity of the active speciesversus the total protein concentration used to determine IC₅₀s.

EXAMPLE 5 VLS Mutants Suitable for DT-Fusion Protein-VLS Modified DTIL-2

1(a) Cytoxocity Assays on Crude Extracts of DT387linker EL-2 VLSMutants.

The DT387 construct was initially used to demonstrate that VLS-modifiedtoxophores could be chemically coupled to a number of targeting ligandsand yield functional targeted toxins. The large-scale production oftargeted toxins following chemical conjugation, however, was not acommercially viable enterprise and the advent of single chain fusionstoxins as exemplified by DT387linker IL-2 circumvents the scale-uppurification problems typically encountered in the development ofconjugate toxins. Fusion toxins, however, do present challenges in thatthe single chain molecules must be purified into an active,appropriately folded form capable of effective delivery of the catalyticdomain of the toxin to targeted cells. Thus, the site-directed changesin VLS modified DT387 and DT387linker IL-2 might not yield functionalmolecules or molecules that can be readily refolded into active fusiontoxins. To confirm the effects of the engineered changes, a number ofVLS modified DT387IL-2 fusion toxins were produced and tested incytotoxicity assays.

Conservative amino acid substitutions in the C and T domains of DT havebeen created. To determine that the changes do not yield inactivetoxophores incapable of producing fusion toxins, cytotoxicity assayswere performed. Readily apparent patterns have emerged which dictate thetype of amino acid substitutions that can be accepted at each of thethree VLS motifs within DT. Results indicate that mutations of the VLSsequences present at amino acid residues 7-9 or 290-292 of the DTtoxophore resulted in less binding to human umbilical vein cellmonolayers in culture. Some constructs demonstrated low levels ofexpression. Consequently additional VLS mutants were developedincluding: V7S, D8E, D8S and D291E.

The cytotoxicities of crude extracts of wild type DAB₃₈₉IL-2, two of theVLS mutants and a control were assayed as indicated. The results arereported as a percentage of control incorporation (no toxin added tocells).

These mutants were incorporated alone or in combination (D8S, V29A andV7A, V29A, I290A variants) into DT387linker IL-2 and have been tested aspartially purified extracts in cytotoxicity assays and results indicatethey are cytotoxic when compared to the negative control, DAB389linkerEGF control, (which contains a targeting ligand to a receptor notexpressed on HUT102/6TG cells) and DAB389linker IL-2. All VLS modifiedmutant toxophore fusion toxins were compared to DAB389linker IL-2produced and tested at similar levels of purity and concentration. Thetriple mutant, DT387(V7A,V29A,D291E)linker IL-2 was expressed infull-length form, despite the valine to alanine change at position 7,and was also cytotoxic. FIG. 13 shows the representative results of acytotoxicity assay using DT387linker IL-2, DT387(D8SV29A)linker IL-2,DT387, DT387(V7AV29A)linker IL2, DT387(V7AV29AI290A)linker IL2,DT387(V7SV29A)linker IL2, and DT387(D8EV29A)linker IL2.

Cytotoxicity assays are performed using HUT102/6TG cells, a human HTLV1transformed T-cell line that expresses high affinity Interleukin-2receptors. HUT102/6TG cells are maintained in RPMI 1640 (Gibco) mediasupplemented with 10% fetal bovine serum, 2 mM glutamine, 50 IU/mlpenicillin and 50 ug/ml streptomycin. The cells are seeded at a densityof 5×10⁴/well into 96 well, V-microtiter plates. The fusion proteintoxins are typically added to the wells in molarities ranging from 10⁻⁷M down to 10⁻¹² M. Final volume in the wells is 2004 The plates areincubated for 18 hours, at 37° C. in a 5% CO₂ environment. The platesare subjected to centrifugation to pellet the cells, the media removedand replaced with 200 μl leucine-free, minimal essential mediumcontaining 1.0 μCi/ml[¹⁴C] leucine (<280 mCi/mmol, Amersham-Pharmacia)and 21 mM glutamine, 50 IU/ml penicillin and 50 μg/ml streptomycin. Thecells are pulsed for 90 minutes and then the plates subjected tocentrifugation to pellet the cells. The supernatant is removed and thecells are lysed in 60 μl, 0.4 M KOH followed by a 10 minute incubationat room temperature. 140 μl of 10% TCA is then added to each well andanother 10 minute, room temperature incubation is performed. Theprecipitated proteins are collected on glass fiber filters using a “PHDcell harvester” and the incorporated radioactivity is determined usingstandard methods. The results are reported as a percentage of control(no fusion protein added to inhibit protein synthesis) [¹⁴C]-leucineincorporation.

Pharmaceutical grade GMP purified DAB₃₈₉IL-2 produced from E. Colitypically yields an IC₅₀ of between 5×10⁻¹¹ M to 1×10⁻¹² M. Partiallypurified toxins exhibit activity between 10-100 fold lower in partiallypurified non-homogenous extracts. Pharmaceutical grade toxins arepurified to homogeneity and the active fractions of refolded fusiontoxins are used as biologically active drug. In the example above weutilize a moderate through put analysis to determine the receptorspecific cytotoxicity of partially purified VLS modified DT-IL-2 fusiontoxins and compared them to the activity of similarly purifiedDAB₃₈₉IL-2. These assays demonstrate comparable activity of the VLSmodified DT387linker BL-2 fusion to DAB₃₈₉IL-2. It should be noted thatthe calculation of specific cytotoxicity was based upon the total amountof protein in the samples of partially fusion toxin. For assaysequimolar concentrations of fusion toxins were tested. As shown below inpanel FIG. 14 panels A and B each fusion toxins construct displayedpatterns on 10% SDS PAGE and Western (anti diphtheria toxin) analysis.In FIG. 14, A is coomassie stained gel of partially purified inclusionbody preparations. Lane 1, molecular weight markers; lane 2, DAB₃₈₉IL-2;lane 3, DT387(V7AV29A)linker IL-2, lane 4, DT387(V7S,V29A)linker IL-2,lane 5, DT387(D8E,V29A) linker EL-2; lane 6, DT387(D8S,V29A) linkerIL-2; lane 7, DT387(V7A,V29A,D291E) linker IL-2. B is correspondingWestern blot with horse anti-DT first antibody and rabbit anti-horsesecondary antibody. The relative amounts of non-fusion toxins protein ineach sample could artificially alter the IC₅₀ of any given construct.That is, the presence of non full length, or non fusion toxin protein inthe samples used in this analysis could potentially account for smalldifferences in IC₅₀.

The cytotoxicity data clearly demonstrate that the modifications thatreduce HUVEC binding can be employed to create functional DTIL-2 fusiontoxins.

Purified DAB₃₈₉ IL-2 produced in E. coli typically yields an IC₅₀ ofbetween 5×10⁻¹¹ M to 1×10⁻¹² M. In the example above, a moderate throughput cytotoxicity assay was used to analyze crude purifications of VLSmodified DT-IL-2 fusion toxins and compared them to the activity ofsimilarly purified DT387linkerIL-2. Insert figure for comparison ofrelative purity of IL2 fusion toxins in this assay.

It should be noted that there is one (x)D(y) motif in IL-2 located atresidues 19-21 (LDL). The contribution of IL-2 to VLS can be determinedby modifying the (x)D(y) motif in the IL-2 and test the modified proteinusing the cytotoxicity assay described above. [For example, usingVLS-modified DT mutants derived from both DT387 and DT387linker IL-2, itis possible to distinguish between effects of the VLS mutations oncatalytic activity, VLS activity and effective delivery of the targetedtoxin to the cytosol of target cells]. The comparison betweenVLS-modified DT mutants of DT387 and DT387linker EL2 will also separatethe effects of VLS sequences of the toxophore alone from the EL-2targeting ligand present in DT387linkerIL-2.

Table 4 summarizes the IC₅₀₅ of VLS-modified DT mutants. Mutants nottested are indicated by “n.t.” Primary screening of mutants wasperformed following expression and crude primary inclusion bodypurification. Complete purification was not performed and the VLSmodified toxophores have all been tested in the context of at least onefusion toxin (EGF receptor or IL-2 receptor targeted) and compared toDT387 based parental fusion toxin expressed and prepared to a similarlevel of purification.

TABLE 4 IC_(50S) of VLS-Modified DT Mutants SINGLE DOUBLE TRIPLE MUTANTMUTANT MUTANT DT387(V7A) >10⁻⁷ DT387(V7A, V29A) >10⁻⁷ DT387 (V7A, V29A,D291E) >10⁻⁷ DT387(D8S) DT387(V7S, V29A) 2 × 10⁻⁸ 2 × 10⁻⁸ DT387(ΔD8)>10⁻⁷ DT387(D8S, V29A) 2.5 × 10⁻¹⁰ DT387(V29A) 2 × 10⁻¹⁰ DT387(D8S,D291S) n.t. DT387(D8E, V29A) 5 × 10⁻⁹ DT387(V29A, D291E) 2 × 10⁻⁹

The IC_(50S) were determined in the cytotoxicity assay as described inExamples 4 and 5, IC_(50S) for DT387linker EGF and DT387linker IL-2 werefound to be in a similar range from 5×10⁻⁹ to 1×10⁻¹⁰ M. Thecytotoxicity of both the parental DAB 389-based fusion toxins andVLS-modified DT387 fusion toxins increased with increasing levels ofpurification. For example pharmaceutical grade DAB289EGF exhibits anIC₅₀ of 4.5×10⁻¹¹ M in these assays whereas crude inclusion bodypreparations of DT387(V29A)linker EGF exhibit an IC₅₀ of 2×10⁻¹⁰ M.

Among the VLS-modified DT387 toxophore constructs tested thus far,DT387(V29A) and DT387(D8S, V29A) appear to maintain cytotoxicitycomparable to wild type. The DT387(D8S) single mutant was not ascytotoxic as the corresponding double mutant indicating the additionalchange to V29A helped stabilize the molecule.

The preferred embodiments of the compounds and methods of the presentinvention are intended to be illustrative and not limiting.Modifications and variations can be made by persons skilled in the artin light of the above teachings specifically those that may pertain toalterations in the DT toxophore surrounding the described VLS sequencesthat could result in reduced HUVEC binding while maintaining near nativefunctionally with respect to the ability to use as a DT toxophore inprotein fusion toxin constructions. It is also conceivable to oneskilled in the art that the present invention can be used for otherpurposes, including, for example, the delivery of other novel moleculesto a selected cell population. It is envisioned that the presentinvention would be employed under those circumstances in which amountsof DT toxophore would be used to deliver such agents in a clinicalsetting or in settings where it would be desirable to reduce as much aspossible the potential for VLS. In this setting the catalytic domain orsome portion thereof would be replaced, or rendered inactive and fusedwith the desired agent or molecule. Acid sensitive or protease sensitivecleavage sites could be inserted between the remnant of the catalyticdomain and the desired agent or molecule. Agents or molecules that mightbe coupled to VLS modified DT toxophore such as disclosed herein includebut are not limited to; peptides or protein fragments, nucleic acids,ogligonucleotides, acid insensitive proteins, glycoproteins, proteins ornovel chemical entities that required selective delivery. Therefore, itshould be understood that changes may be made in the particularembodiments disclosed which are within the scope of what is described asdefined by the appended claims.

What is claimed is:
 1. An isolated polypeptide comprising amino acidresidues 1-380 of SEQ ID NO:4, wherein the polypeptide comprises aminoacid substitution at position I290 or D291 of SEQ ID NO:4, and,optionally with at least one amino acid substitution or deletion madewithin the regions selected from the group consisting of residues 7-9,29-31 and 290-292, and wherein said DT variant has cytotoxicitycomparable to that of a DT molecule having a sequence of SEQ ID NO:4. 2.The isolated polypeptide of claim 1, wherein the polypeptide has reducedbinding activity to human vascular endothelial cells (HUVECs) comparedto a polypeptide comprising an amino acid sequence as recited in SEQ IDNO:4 without substitutions.
 3. The isolated polypeptide of claim 1,wherein the polypeptide comprises an optional substitution at amino acidresidue V29 selected from V29A, V29D or V29I.
 4. The isolatedpolypeptide of claim 1, wherein the optional substitution is V7A, V7S,DBE, DBS, I290A, D291E or D291S or any combination thereof.
 5. Theisolated polypeptide of claim 1, further comprising a protein selectedfrom the group consisting of EGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, INFα, INFγ,GM-CSF, G-CSF, M-CSF, TNF, EGF, VEGF, Ephrin, BFGF and TGF.
 6. Theisolated polypeptide of claim 5, selected from the group consisting ofEGF, IL-1, IL-2, IL-3, and IL-7.
 7. The isolated polypeptide of claim 1,wherein the optional substitution is V7AV29A.
 8. The isolatedpolypeptide of claim 1, wherein the substitutions are V7V29D291 orV7V29I290.
 9. The isolated polypeptide of claim 1, wherein thesubstitutions are V7AV29AI290A or V7AV29AD291E.
 10. A compositioncomprising the isolated polypeptide of claim 1 in a pharmaceuticallyacceptable carrier.
 11. The isolated polypeptide of claim 1, furthercomprising a linker peptide moiety at amino acid residue 380 of SEQ IDNO:4.
 12. The isolated polypeptide of claim 11, wherein the linkerpeptide moiety comprises non-charged amino acid residues.
 13. Theisolated polypeptide of claim 11, wherein the linker peptide comprisesthe sequence as set forth in SEQ ID NO:5.
 14. The isolated polypeptideof claim 13, further comprising EGF on the C-terminus of the protein.